Due to the development of gene recombination technology, industrially useful polypeptides have been industrially produced. In particular, a lot of antibody pharmaceutical products and protein pharmaceutical products have been developed in the field of bio-pharmaceutical products.
Such an industrially useful polypeptide is often produced by culturing recombinant host cells transfected with a vector containing a gene encoding the polypeptide to be produced, and a technique for purifying the desired polypeptide from the culture solution is indispensable.
A typical step of purifying a polypeptide comprises a step of separating cells from a culture solution, and further comprises multiple steps in combination for removing cell fragments, unnecessary polypeptides, organic substances, inorganic substances or salts, and the like, in the culture solution from which the cells were removed.
In recent years, the cultivation technique has developed considerably, and therefore, in a culture solution, cells or cell fragments are present at a high density, and a polypeptide to be produced is present at a high density. As a result, in a step of separating the cells from the culture solution by utilizing a centrifuge or a membrane, a method for improving the efficiency, for example, the clogging of a membrane is reduced, or the like is demanded.
For example, a procedure called “acid precipitation”, in which unnecessary substances in a culture solution are precipitated in advance by adding an acid to the culture solution when the cultivation is completed, is one of the methods of improving the efficiency of the step of separating cells from the culture solution immediately after the procedure (Non-Patent Document 1).
Further, the purification step should be capable of not only providing a polypeptide with a purity suitable for use, but also providing a high yield by preventing the denaturation or loss of the polypeptide during purification. Further, the purification step is desirably configured to comprise steps in an efficient order so as to make use of the effect of the respective steps.
Typical denaturation of a polypeptide during purification includes the reduction of a disulfide bond of a polypeptide, the cleavage of a peptide bond, the association of polypeptides, and the like before or after removing cells from the culture solution. It is known that in particular, a thioredoxin system is involved in the reduction of a disulfide bond of a polypeptide in some cases (Non-Patent Documents 2, 3, and 4).
This thioredoxin system is essentially intracellularly present in the host cells which produce a polypeptide. However, in the production of a polypeptide, during cultivation of recombinant host cells, due to apoptosis or the like of the cells, after cultivation of the recombinant host cells, cell membranes of the recombinant host cells are ruptured by mechanical stimulation through each procedure of the purification step, and therefore, thioredoxin, thioredoxin reductase, glucose-6-phosphate dehydrogenase, a hexokinase, and substrates therefor, which are the constituent elements of the thioredoxin system, are released in the culture solution.
Therefore, it is considered that in the production of a polypeptide, the thioredoxin system is also present in the culture solution, and this can be a cause of the reduction of a polypeptide to be produced.
Examples of a conventional method for preventing the reduction of a disulfide bond in which this thioredoxin system is involved include a method of adding aurothioglucose (ATG) which is a thioredoxin inhibitor or ethylenediaminetetraacetic acid (EDTA) which inhibits a hexokinase to a culture solution after culturing recombinant host cells and before separating the cells or after separating the cells.
Additional examples thereof include a method of depleting glucose-6-phosphate (G6P) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) by filling with air in the culture solution after recombinant host cells are cultured and the cells are separated, and the like (Patent Document 1).